Anticancer Activity of the Bark Extract of Phyllanthus emblica on Cholangiocarcinoma In Vitro

Main Article Content

Papavee Samatiwat
Pongsathorn Chankhonkaen
Yamaratee Jaisin
Piyanee Ratanachamnong
Cholticha Niwaspragrit
Ruttachuk Rungsiwiwut
Boonchua Dhorranintra


Cholangiocarcinoma (CCA) is the most common biliary epithelial malignancy in Northeast Thailand. The critical obstacle of CCA therapy is cancer chemotherapy resistance with intolerance of adverse drug reactions. A new approach has been the investigation of alternative herbal medicines for cancer therapy. The fruit of Phyllanthus emblica (PE) has several pharmacological properties, including potent anticancer properties. The present study aimed to evaluate the antiproliferative activity of the bark extract of Phyllanthus emblica in methanol on the KKU-452 CCA cell line. The cytotoxicity of Thai herbal plants, Solanum torvum (fruit), Nephelium hypoleucum (fruit), Schleichera oleosa (fruit), Antidesma thwaitesianum (fruit), Tamarindus indica (fruit, leaf, bark) and Phyllanthus emblica (fruit, leaf, bark), were screened against KKU-452. Cell proliferation, apoptosis and migration were evaluated using MTT-, Annexin V apoptosis- and wound healing assays, respectively. Reactive oxygen species (ROS) was determined by DCFH-DA fluorogenic dye staining.  Bark of PE extracts was subjected to HPLC analysis. Results showed that the bark extract of P. emblica only had a potent cytotoxic effect on KKU-452 (IC50 of 52.2 µg/ml) and significant induction of apoptosis.  Other plant extracts showed low potency of cytotoxic effects. Cell migration was significantly inhibited by P. emblica at 25 and 50 µg/ml by 42.8 and 32.9%, respectively.  Moreover, the extract at 50 µg/ml induced oxidative stress via ROS production at 31% when compared with non-treated cells. Phenolic acids and flavonoids are the important phytochemical components in the bark extract of P. emblica. The cytotoxic effect of the bark extract of P. emblica was related to free radical generation.

Article Details

Research Articles


Sripa B, Pairojkul C. Cholangiocarcinoma: lessons from Thailand. Curr Opin Gastroenterol. 2008;24(3):349-356.

Khuntikeo N, Chamadol N, Yongvanit P, Loilome W, Namwat N, Sithithaworn P, et al. Cohort profile: cholangiocarcinoma screening and care program (CASCAP). BMC Cancer. 2015;15:459.

Kamsa-Ard S, Luvira V, Suwanrungruang K, Kamsa-Ard S, Luvira V, Santong C, et al. Cholangiocarcinoma trends, incidence, and relative survival in Khon Kaen, Thailand from 1989 through 2013: A population-based cancer registry study. J Epidemiol. 2019;29(5):197-204.

Ong CK, Subimerb C, Pairojkul C, Wongkham S, Cutcutache I, Yu W, et al. Exome sequencing of liver fluke-associated cholangiocarcinoma. Nat Genet. 2012;44(6): 690-693.

Valle J, Wasan H, Palmer DH, Cunningham D, Anthoney A, Maraveyas A, et al. Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N Engl J Med. 2010; 362(14):1273-1281.

Yang L, Jiang JG, Li WF, Chen J, Wang DY, Zhu L. Optimum extraction process of polyphenols from the bark of Phyllanthus emblica L. based on the response surface methodology. J Sep Sci. 2009;32(9):1437-1444.

Kittiratphatthana N, Kukongviriyapan V, Prawan A, Senggunprai L. Luteolin induces cholangiocarcinoma cell apoptosis through the mitochondrial-dependent pathway mediated by reactive oxygen species. J Pharm Pharma-col. 2016;68(9):1184-1192.

Aneknan P, Kukongviriyapan V, Prawan A, Kongpetch S, Sripa B, Senggunprai L. Luteolin arrests cell cycling, induces apoptosis and inhibits the JAK/STAT3 pathway in human cholangiocarcinoma cells. Asian Pac J Cancer Prev. 2014;15(12):5071-5076.

Senggunprai L, Thammaniwit W, Kukong-viriyapan V, Prawan A, Kaewseejan N, Siriamornpun S. Cratoxylum formosum extracts inhibit growth and metastasis of cholangiocarcinoma cells by modulating the NF-κB and STAT3 pathways. Nutr Cancer. 2016;68(2):328-341.

Zhang J, Su G, Tang Z, Wang L, Fu W, Zhao S, et al. Curcumol exerts anticancer effect in cholangiocarcinoma cells via down-regulating CDKL3. Front Physiol. 2018;9:234.

Qin Y, Cui W, Yang X, Tong B. Kaempferol inhibits the growth and metastasis of cholangiocarcinoma in vitro and in vivo. Acta Biochim Biophys Sin (Shanghai). 2016;48(3):238-245.

Promraksa B, Phetcharaburanin J, Namwat N, Techasen A, Boonsiri P, Loilome W. Evaluation of anticancer potential of Thai medicinal herb extracts against cholangio-carcinoma cell lines. PLoS One. 2019;14(5): e0216721.

Panigrahi S, Muthuraman M, Natesan R, Brindha P. Anticancer activity of ethanolic extract of Solanum torvum SW. Int J Pharm Pharm Sci. 2014;6:93-98.

Chusri S, Singthong P, Kaewmanee T. Antioxidant, anticancer, and cytotoxic effects of Thai traditional herbal preparations consumed as rejuvenators. CyTA J Food. 2015;13(1):40-48.

Shahrajabian MH, Sun W, Cheng Q. Modern pharmacological actions of Longan fruits and their usages in traditional herbal remedies. J Med Plants Stud. 2019;7:179-185.

Lin CC, Chung YC, Hsu CP. Potential roles of longan flower and seed extracts for anticancer. World J Exp Med. 2012;2(4):78-85.

Bhatia H, Kaur J, Nandi S, Gurnani V, Chowdhury A, Reddy PH, et al. A review on Schleichera oleosa: Pharmacological and environmental aspects. Pharm Res. 2013; 6(1):224-229.

Aravind SR, Joseph MM, Varghese S, Balaram P, Sreelekha TT. Antitumor and immunopotentiating activity of polysaccharide PST001 isolated from the seed kernel of Tamarindus indica: an in vivo study in mice. ScientificWorld Journal. 2012;2012:361382.

Liu X, Zhao M, Wu K, Chai X, Yu H, Tao Z, et al. Immunomodulatory and anticancer activities of phenolics from emblica fruit (Phyllanthus emblica L.). Food Chemistry. 2012;131(2):685-690.

Bharti AC, Donato N, Aggarwal BB. Curcumin (diferuloylmethane) inhibits constitutive and IL-6-inducible STAT3 phosphorylation in human multiple myeloma cells. J Immunol. 2003;171(7): 3863-3871.

Loo G. Redox-sensitive mechanisms of phytochemical-mediated inhibition of cancer cell proliferation (review). J Nutr Biochem. 2003;14(2):64-73.

Zhao T, Sun Q, Marques M, Witcher M. Anticancer properties of Phyllanthus emblica (Indian Gooseberry). Oxid Med Cell Longev. 2015;2015:950890.

Unander DW, Webster GL, Blumberg BS. Records of usage or assays in Phyllanthus (Euphorbiaceae). I. Subgenera Isocladus, Kirganelia, Cicca and Emblica. J Ethno-pharmacol. 1990;30(3):233-264.

Chaphalkar R, Apte KG, Talekar Y, Ojha SK, Nandave M. Antioxidants of Phyllanthus emblica L. bark extract provide hepato-protection against ethanol-induced hepatic damage: A comparison with silymarin. Oxid Med Cell Longev. 2017;2017:3876040.

Leelawat S, Leelawat K. Molecular mechanisms of cholangiocarcinoma cell inhibition by medicinal plants. Oncol Lett. 2017;13(2): 961-966.

Liu X, Zhao M, Wang J, Yang B, Jiang Y. Antioxidant activity of methanolic extract of emblica fruit (Phyllanthus emblica L.) from six regions in China. J Food Compost Anal. 2008;21(3):219-228.

Al-Rehaily AJ, Al-Howiriny TA, Al-Sohaibani MO, Rafatullah S. Gastroprotective effects of 'Amla' Emblica officinalis on in vivo test models in rats. Phytomedicine. 2002;9(6): 515-522.

Nosal'ova G, Mokry J, Hassan KM. Antitussive activity of the fruit extract of Emblica officinalis Gaertn. (Euphorbiaceae). Phytomedicine. 2003;10(6-7):583-589.

Jose JK, Kuttan R. Hepatoprotective activity of Emblica officinalis and Chyavanaprash. J Ethnopharmacol. 2000 Sep;72(1-2):135-140.

Nain P, Saini V, Sharma S, Nain J. Antidiabetic and antioxidant potential of Emblica officinalis Gaertn. leaves extract in streptozotocin-induced type-2 diabetes mellitus (T2DM) rats. J Ethno-pharmacol. 2012;142(1):65-71.

Mahata S, Pandey A, Shukla S, Tyagi A, Husain SA, Das BC, et al. Anticancer activity of Phyllanthus emblica Linn. (Indian goose-berry): inhibition of transcription factor AP-1 and HPV gene expression in cervical cancer cells. Nutr Cancer. 2013;65 Suppl 1:88-97.

Zhu X, Wang J, Ou Y, Han W, Li H. Polyphenol extract of Phyllanthus emblica (PEEP) induces inhibition of cell proliferation and triggers apoptosis in cervical cancer cells. Eur J Med Res. 2013;18:46.

Sumalatha D. Antioxidant and antitumor activity of Phyllanthus emblica in colon cancer cell lines. Int J Curr Microbiol Appl Sci 2013;2:189-195.

Talekar Y, Das B, Paul T, Talekar D, Apte K, Parab P. Wound healing activity of aqueous and ethanolic extract of bark of Emblica officinalis in Wistar rats Inventi Impact: Planta Activa. 2012;4:1-5.

Samatiwat P, Tabtimmai L, Suphakun P, Jiwacharoenchai N, Toviwek B, Kukong-viriyapan V, et al. The effect of the EGFR - targeting compound 3-[(4-phenylpyrimidin-2-yl) amino] benzene-1-sulfonamide (13f) against cholangiocarcinoma cell lines. Asian Pac J Cancer Prev. 2021;22(2):381-390.

Cai Y, Zhang J, Chen NG, Shi Z, Qiu J, He C, et al. Recent advances in anticancer activities and drug delivery systems of tannins. Med Res Rev. 2017;37(4):665-701.

Saensa-Ard S, Leuangwattanawanit S, Senggunprai L, Namwat N, Kongpetch S, Chamgramol Y, et al. Establishment of cholangiocarcinoma cell lines from patients in the endemic area of liver fluke infection in Thailand. Tumour Biol. 2017; 39(11):1010428317725925.

Samatiwat P, Prawan A, Senggunprai L, Kukongviriyapan U, Kukongviriyapan V. Nrf2 inhibition sensitizes cholangiocarcinoma cells to cytotoxic and antiproliferative activities of chemotherapeutic agents. Tumour Biol. 2016; 37(8):11495-11507.

Gomes A, Fernandes E, Lima JL. Fluorescence probes used for detection of reactive oxygen species. J Biochem Biophys Methods. 2005; 65(2-3):45-80.

Sarkar R, Kundu A, Banerjee K, Saha S. Anthocyanin composition and potential bioactivity of karonda (Carissa carandas L.) fruit: An Indian source of biocolorant. LWT. 2018;93:673-678.

Ravishankar D, Rajora AK, Greco F, Osborn HM. Flavonoids as prospective compounds for anticancer therapy. Int J Biochem Cell Biol. 2013;45(12):2821-2831.

Wahle KW, Brown I, Rotondo D, Heys SD. Plant phenolics in the prevention and treatment of cancer. Adv Exp Med Biol. 2010;698:36-51.

Poon IKH, Hulett MD, Parish CR. Molecular mechanisms of late apoptotic/necrotic cell clearance. Cell Death Differ. 2010;17(3): 381-397.

Machana S, Weerapreeyakul N, Barusrux S, Nonpunya A, Sripanidkulchai B, Thiti-metharoch T. Cytotoxic and apoptotic effects of six herbal plants against the human hepatocarcinoma (HepG2) cell line. Chin Med. 2011;6(1):39.

Kalinina TS, Bannova AV, Dygalo NN. Quantitative evaluation of DNA fragmentation. Bull Exp Biol Med. 2002;134(6):554-556.

Khan M, Qais F, Ahmad I. Indian berries and their active compounds: Therapeutic Potential in Cancer Prevention. New Look to Phyto-medicine 2019.179-201.

Jose JK, Kuttan G, Kuttan R. Antitumour activity of Emblica officinalis. J Ethno-pharmacol. 2001;75(2-3):65-69.

Kaur S, Michael H, Arora S, Harkonen PL, Kumar S. The in vitro cytotoxic and apoptotic activity of Triphala--an Indian herbal drug. J Ethnopharmacol. 2005;97 (1):15-20.

Ngamkitidechakul C, Jaijoy K, Hansakul P, Soonthornchareonnon N, Sireeratawong S. Antitumour effects of Phyllanthus emblica L.: induction of cancer cell apoptosis and inhibition of in vivo tumour promotion and in vitro invasion of human cancer cells. Phytother Res. 2010;24(9):1405-1413.

Sandhya T, Lathika KM, Pandey BN, Mishra KP. Potential of traditional ayurvedic formulation, Triphala, as a novel anticancer drug. Cancer Lett. 2006; 231(2):206-214.

Pinmai K, Chunlaratthanabhorn S, Ngamkitidechakul C, Soonthornchareon N, Hahnvajanawong C. Synergistic growth inhibitory effects of Phyllanthus emblica and Terminalia bellerica extracts with conventional cytotoxic agents: doxorubicin and cisplatin against human hepatocellular carcinoma and lung cancer cells. World J Gastroenterol. 2008;14 (10):1491-1497.

Sharma N, Trikha P, Athar M, Raisuddin S. Inhibitory effect of Emblica officinalis on the in vivo clastogenicity of benzo[a]pyrene and cyclophosphamide in mice. Hum Exp Toxicol. 2000;19(6): 377-384.

Lu Y, Jiang F, Jiang H, Wu K, Zheng X, Cai Y, et al. Gallic acid suppress cell viability, proliferation, invasion and angiogenesis in human glioma cells. Eur J Pharmacol. 2010 Sep 1;641(2-3):102-107.

Wang Z, Zhou J, Tang Y, Shi H, Huang XW, Deng JH, et al. Quercetin induces P53-independent G2/M arrest and apoptosis in cancer cells. Chinese J. Pharmacol. Toxicol. 2018;32:790-796.

Chou CC, Yang JS, Lu HF, Ip SW, Lo C, Wu CC, et al. Quercetin-mediated cell cycle arrest and apoptosis involving activation of a caspase cascade through the mitochondrial pathway in human breast cancer MCF-7 cells. Arch Pharm Res. 2010;33(8):1181-1191.

Seo HS, Ku JM, Choi HS, Choi YK, Woo JK, Kim M, et al. Quercetin induces caspase-dependent extrinsic apoptosis through inhibition of signal transducer and activator of transcription 3 signaling in HER2-overexpressing BT-474 breast cancer cells. Oncol Rep. 2016;36(1):31-42.

Lee LT, Huang YT, Hwang JJ, Lee PP, Ke FC, Nair MP, et al. Blockade of the epidermal growth factor receptor tyrosine kinase activity by quercetin and luteolin leads to growth inhibition and apoptosis of pancreatic tumor cells. Anticancer Res. 2002;22(3):1615-1627.

Bhat FA, Sharmila G, Balakrishnan S, Arunkumar R, Elumalai P, Suganya S, et al. Quercetin reverses EGF-induced epithelial to mesenchymal transition and invasiveness in prostate cancer (PC-3) cell line via EGFR/PI3K/Akt pathway. J Nutr Biochem. 2014;25(11):1132-1139.

Walker EH, Pacold ME, Perisic O, Stephens L, Hawkins PT, Wymann MP, et al. Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Mol Cell. 2000;6(4):909-919.

Granato M, Rizzello C, Gilardini Montani MS, Cuomo L, Vitillo M, Santarelli R, et al. Quercetin induces apoptosis and autophagy in primary effusion lymphoma cells by inhibiting PI3K/AKT/mTOR and STAT3 signaling pathways. J Nutr Biochem. 2017;41: 124-136.

Naus PJ, Henson R, Bleeker G, Wehbe H, Meng F, Patel T. Tannic acid synergizes the cytotoxicity of chemotherapeutic drugs in human cholangiocarcinoma by modulating drug efflux pathways. J Hepatol. 2007;46(2): 222-229.

Chen LI, Guo Y, Alsaif G, Gao Y. Total Flavonoids isolated from Diospyros kaki L. f. leaves induced apoptosis and oxidative stress in human cancer cells. Anticancer Res. 2020;40(9):5201-5210.

Pal A, Sengupta S, Kundu R. Tiliacora racemosa leaves induce oxidative stress mediated DNA damage leading to G2/M

phase arrest and apoptosis in cervical cancer cells SiHa. J Ethnopharmacol. 2021;269: 113686.

Macri R, Musolino V, Gliozzi M, Carresi C, Maiuolo J, Nucera S, et al. Ferula L. plant extracts and dose-dependent activity of natural sesquiterpene ferutinin: From antioxidant potential to cytotoxic effects. Molecules. 2020;25(23):5768.

Jung EM, Lim JH, Lee TJ, Park JW, Choi KS, Kwon TK. Curcumin sensitizes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through reactive oxygen species-mediated upregulation of death receptor 5 (DR5). Carcinogenesis. 2005;26(11):1905-1913.